Several studies have shown that the measured potency of factor VIII (FVIII) products can vary depending on the test used to assay the products. In the case of one recombinant FVIII (rFVIII), a patient will receive 30% fewer moles of FVIII if a chromogenic assay is used, rather than a one-stage clotting test. In contrast, the plasma clotting assay gives about 1.25 fold higher potency than the chromogenic assay when applied to another rFVIII product that lacks a B-domain segment of FVIII. The chromogenic test is mandated in Europe, while the one stage clotting assay is the standard used in the United States. Understanding the reason for assay discrepancies will help to determine the feasibility of accepting a single potency test for factor VIII. Results: 1) Structural comparison of factor VIII products. The rFVIII product that had the most divergent potency results when tested by the two assays, had more polypeptide species that contained the B-domain, than plasma-derived FVIII. In addition, the product contained a >200 kDa band not observed in plasma-derived FVIII. Antibody mapping suggests that the >200 kDa polypeptide is an N-terminal truncated single polypeptide form of FVIII. We are investigating the effect of these differences on the potency assays. 2) Investigation of components that affect FVIII activation. Thrombin activates FVIII by cleaving several peptide bonds located on the heavy and light chains of FVIII. To evaluate the importance of the B-domain and acidic regions of FVIII, on activation of FVIII by thrombin, we are evaluating the binding sites on thrombin for FVIII. a-Thrombin, blocked at its active site, inhibited thrombin activation of FVIII, suggesting that a site(s) on thrombin, other than its active site, is needed for FVIII activation. Thrombin-catalyzed cleavages of both the heavy and light chains of FVIII were inhibited by a sulfated polypeptide segment of the FVIII heavy chain (F8II). F8II did not inhibit the amidolytic activity of thrombin. These findings further support the involvement of sites on thrombin, other than its active site, in FVIII activation. To investigate whether the site might be the same as the fibrinogen-recognition exosite of thrombin, the C-terminal dodecapeptide of hirudin, which binds to the exosite, was tested as an inhibitor of thrombin proteolysis of FVIII. The peptide did not inhibit thrombin cleavage of FVIII. F8II did not inhibit thrombin digestion of fibrinogen. Furthermore, g-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII. We conclude that there is a novel recognition site on thrombin for FVIII, that is different from the fibrinogen-recognition exosite, and the active site. An acidic region of FVIII binds to a site on thrombin that is involved in the rate-limiting step of FVIII activation.